DNA-looping and enhancer activity: association between DNA-bound NtrC activator and RNA polymerase at the bacterial glnA promoter.

Abstract

The NtrC protein activates transcription of the ginA operon of enteric bacteria by stimulating the formation of stable "open" complexes by RNA polymerase (oMholoenzyme form). To regulate the ginA promoter, NtrC binds to sites that have the properties of transcriptional enhancers: the sites will function far from the promoter and in an orientation-independent fashion. To investigate the mechanism of enhancer function, we have used electron microscopy to visualize the interactions of purified NtrC and RNA polymerase with their DNA binding sites and with each other. Under conditions that allow the formation of open complexes, about 30% of DNA molecules carry both RNA polymerase and NtrC bound to their specific sites. Of these, about 15% form looped structures in which NtrC and the RNA polymerase-promoter complex are in contact. The length of the looped DNA is that predicted from the spacing that was engineered between the enhancer and the ginA promoter (390 base pairs). As expected for activation intermediates, the looped structures disappear when RNA polymerase is allowed to transcribe the DNA. We conclude that the NtrC enhancer functions by means of a direct association between DNA-bound NtrC and RNA polymerase (DNA-looping model). Association of DNA-bound proteins appears to be the major mechanism by which different types of site-specific DNA transactions are localized and controlled.Enhancer sequences have been defined by their function in activating transcription from relatively long distances in an orientation-independent manner (1, 2). There are many wellcharacterized examples of enhancers controlling eukaryotic cellular and viral genes (3-5). Although initially defined in eukaryotes, regulation of transcription from distant sites has been observed for many prokaryotic promoters (6, 7). The site for positive regulation of the glnA operon of enteric bacteria is a particularly well-defined example of a prokaryotic enhancer (8-10). To investigate the mechanism of enhancer action, we have studied the activation of transcription from the glnA promoter by the NtrC enhancer-binding protein.Three principal models have been proposed for enhancer action (11-13). In the first, the regulatory protein (or RNA polymerase) associates with the DNA at the enhancer site and then traverses the DNA to the start site for RNA synthesis (entry-site or scanning model) (1,14). In the second model, the enhancer-binding protein facilitates transcription by initiating a change in DNA structure that is propagated from the enhancer site to the promoter (e.g., a site-specific DNA topoisomerase) (15). In the third model, the enhancerbound regulatory protein stimulates transcription by a direct protein-protein interaction with RNA polymerase at the promoter site (or with other proteins that contact polymerase) (DNA-looping model) (11-13, 16).The DNA-looping model for enhancer action has been considered attractive for several reasons. The interaction between DNA-bound proteins has been for some time an established principle for contr...

The Spatial Organization of the VirR Boxes Is Critical for VirR-Mediated Expression of the Perfringolysin O Gene, pfoA , from Clostridium perfringens

“…Evidence for this interaction was obtained in gel mobility shift assays, in which VirR bound to the VirR boxes was required for the efficient and specific binding of CpRNAP to the pfoA promoter. These results are in agreement with those of other studies that have shown that response regulators can interact with components of RNA polymerase (20,26,45,46).…”

“…Alternatively, activators such as NtrC (24) and NifA (10) stimulate the isomerization of bound RNA polymerase from a closed complex to an open complex (29). It is unlikely that VirR would act in the same fashion as NtrC and NifA, as these proteins bind well upstream of the promoter (9,46). In contrast, the VirR boxes are located immediately upstream of the Ϫ35 box of the pfoA promoter region.…”

A conserved region in the σ54‐dependent activator DctD is involved in both binding to RNA polymerase and coupling ATP hydrolysis to activation

“…The activator converts the closed promoter complex to an open complex that is transcriptionally competent (Sasse-Dwight and Gralla, 1988;Morett and Buck, 1989;Popham et al, 1989;Lee et al, 1993;Lee et al, 1994;Perez-Martin and de Lorenzo, 1996b). To catalyse this isomerization, the activator must hydrolyse ATP (Weiss et al, 1991;Lee et al, 1993;Lee et al, 1994;Perez-Martin and de Lorenzo, 1996b) and make productive contact with 54 -holoenzyme through a DNA loop (Buck et al, 1987;Su et al, 1990;Wedel et al, 1990). Interactions between the activator and 54 -holoenzyme are transient, and sites involved in proteinprotein interaction have not been identified in these proteins.…”

Probing the assembly of transcription initiation complexes through changes in σ N protease sensitivity

“…Isomerization of the closed complex to transcriptionally competent open complexes requires an activator protein bound to a DNA sequence with enhancer-like properties (7,8) and nucleoside triphosphate hydrolysis by the activator protein (5,9,10). Activation appears to involve direct contact between activator and the holoenzyme (11)(12)(13).…”